1. Field of the Invention
The present invention relates to a protein which binds to a stress responsive element and regulates the transcription of genes located downstream of the element; a gene coding for the above protein; a recombinant vector comprising the gene; a transformant comprising the recombinant vector; a transgenic plant comprising the gene; a method for producing the above protein using the transformant; and a method for determining a stress level in a plant.
2. Prior Art
Transcription of genes is performed by RNA polymerase. RNA polymerase synthesizes ribonucleoside phosphates in the 5xe2x80x2 to 3xe2x80x2 direction using double-stranded DNA as a template in a primer independent manner. In the case of Escherichia coli, for example, its RNA polymerase takes the form of a holoenzyme in which xcfx81 factor having promoter recognition ability is bound to the core enzyme xcex2xe2x80x2xcex2xcex12. This RNA polymerase initiates transcription and elongates RNA chain; the transcription is terminated by the binding of xcfx81 factor. On the other hand, in the case of eucaryotes, RNA polymerase is classified into RNA polymerases I, II and III, any of which has a complicated structure composed of more than 10 subunits. RNA polymerase I selectively transcribes rRNA; RNA polymerase II selectively transcribes mRNA precursor; and RNA polymerase III selectively transcribes tRNA and 5SrRNA. The amount of RNA synthesized by such RNA polymerase varies widely depending on the growth stage of the relevant cells and environmental changes around them. A transcription factor which positively or negatively regulates the transcription initiation of RNA polymerase is deeply involved in the variation in the amount of RNA synthesis.
Generally, living cells are exposed to an external environment composed of a number of factors including temperature, pressure, oxygen, light, radioactive rays, metal ions, organic compounds, etc. When these factors vary, cells perceive such changes as stress and make characteristic responses to them. For example, cells exhibit a response called xe2x80x9cheat shock responsexe2x80x9d to high temperatures. From this response, the expression of a group of heat shock proteins (HSPs) is induced. HSPs prevent the irreversible precipitation of heat-denatured proteins and have the function of molecular chaperone that facilitates the refolding of such proteins, thereby protecting cells from heat stress. It is known that a transcription factor called xe2x80x9cheat shock factor (HSF)xe2x80x9d plays an important role in the manifestation of the above-described heat shock response in human, Xenopus, Drosophila, etc. [Kazuhiro Nagata, Cell Technology, 10:348-356 (1991)]. When activated by heat shock, HSF binds to heat shock element (HSE) located upstream of a gene coding for HSP (also known as heat shock gene) to thereby promote the transcription of the heat shock gene.
On the other hand, it is also reported that plants induce stress proteins such as LEA proteins, water channel proteins or synthetases for compatible solutes in their cells when they are exposed to stress such as dehydration, low temperature, freezing or salt, thereby protecting their cells from such stress. However, much more research is required to elucidate transcription factors which regulate the transcription of genes encoding those stress proteins.
It is an object of the present invention to provide a protein which regulates the transcription of genes located downstream of a stress responsive element which is essential for controlling stress responsive gene expression; a gene encoding the protein; a recombinant vector comprising the gene; a transformant comprising the recombinant vector; a transgenic plant comprising the gene; a method for producing the above protein using the transformant; and a method for determining a stress level in a plant.
As a result of extensive and intensive researches toward the solution of the above-described problem, the present inventors have succeeded in isolating from a low temperature resistant plant Arabidopsis thaliana a gene coding for a transcription factor which binds to a stress responsive element and activates the transcription of genes located downstream of the element. Thus, the present invention has been achieved.
The present invention relates to the following recombinant protein (a) or (b):
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10;
(b) a protein which consists of the amino acid sequence having deletion, substitution or addition of at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10 and which regulates the transcription of genes located downstream of a stress responsive element.
Further, the present invention relates to a transcription factor gene coding for the following protein (a) or (b):
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10;
(b) a protein which consists of the amino acid sequence having deletion, substitution or addition of at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10 and which regulates the transcription of genes located downstream of a stress responsive element.
Further, the present invention relates to a gene comprising the following DNA (c) or (d):
(c) a DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9;
(d) a DNA which hybridizes with the DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9 under stringent conditions and which codes for a protein that regulates the transcription of genes located downstream of a stress responsive element.
Specific examples of the above-mentioned stress include dehydration stress, low temperature stress and salt stress.
Further, the present invention relates to a recombinant vector comprising the gene of the invention.
Further, the present invention relates to a transformant comprising the recombinant vector.
Further, the present invention relates to a transgenic plant comprising the gene of the invention.
Further, the present invention relates to a method for producing a protein which regulates the transcription of genes located downstream of a stress responsive element, comprising culturing the above transformant in a medium and recovering the protein from the resultant culture.
Further, the present invention relates to a method for determining a stress level in a plant, comprising determining a transcription level of the gene of the invention in the plant.